Gene therapy provides methods for combating chronic infectious diseases (e.g., HIV infection), as well as non-infectious diseases including cancer and some forms of congenital defects such as enzyme deficiencies. Several approaches for introducing nucleic acids into cells in vivo, ex vivo and in vitro have been used. These include liposome based gene delivery (Debs and Zhu (1993) WO 93/24640 and U.S. Pat. No. 5,641,662); Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691; Rose U.S. Pat No. 5,279,833; Brigham (1991) WO 91/06309; and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414) and adenoviral vector mediated gene delivery, e.g., to treat cancer (see, e.g., Chen et al. (1994) Proc. Nat""l. Acad. Sci. USA 91: 3054-3057; Tong et al. (1996) Gynecol. Oncol. 61: 175-179; Clayman et al. (1995) Cancer Res. 5: 1-6; O""Malley et al. (1995) Cancer Res. 55: 1080-1085; Hwang et al. (1995) Am. J. Respir. Cell Mol. Biol. 13: 7-16; Haddada et al. (1995) Curr. Top. Microbiol. Immunol. 199 (Pt. 3): 297-306; Addison et al. (1995) Proc. Nat""l. Acad. Sci. USA 92: 8522-8526; Colak et al. (1995) Brain Res. 691: 76-82; Crystal (1995) Science 270: 404-410; Elshami et al. (1996) Human Gene Ther. 7: 141-148; Vincent et al. (1996) J. Neurosurg. 85: 648-654). Replication-defective retroviral vectors harboring a therapeutic polynucleotide sequence as part of the retroviral genome have also been used, particularly with regard to simple MuLV vectors. See, e.g., Miller et al. (1990) Mol. Cell. Biol. 10:4239 (1990); Kolberg (1992) J. NIH Res. 4:43, and Cornetta et al. Hum. Gene Ther. 2:215 (1991)).
One of the most attractive targets for gene therapy is HIV infection. The pandemic spread of HIV has driven an intense world-wide effort to unravel the molecular mechanisms and life cycle of these viruses. It is now clear that the life cycle of HIVs provide many potential targets for inhibition by gene therapy, including cellular expression of transdominant mutant gag and env nucleic acids to interfere with virus entry, TAR (the binding site for tat, which is typically required for transactivation) decoys to inhibit transcription and trans activation, and RRE (the binding site for Rev; i.e., the Rev Response Element) decoys and transdominant Rev mutants to inhibit RNA processing. See, Rosenburg and Fauci (1993) in Fundamental Immunology, Third Edition Paul (ed) Raven Press, Ltd., New York and the references therein for an overview of HIV infection and the HIV life cycle. Gene therapy vectors encoding ribozymes, antisense molecules, decoy genes, transdominant genes and suicide genes, including retroviruses are described in Yu et al., Gene Therapy (1994) 1:13-26. Antisense and ribozyme therapeutic agents are of increasing importance in the treatment and prevention of HIV infection.
Despite the various gene therapeutic approaches now underway for treating cancer, HIV and the like, there are a variety of limitations of the delivery systems currently used in gene therapy. For instance, with regard to HIV treatment, the extensively used murine retroviral vectors transduce (transfer nucleic acids into) human peripheral blood lymphocytes poorly, and fail to transduce non-dividing cells such as monocytes/macrophages, which are known to be reservoirs for HIV. New safer vectors for the delivery of viral inhibitors, particularly to non-dividing hematopoietic stem cells for the treatment of HIV infection, are desirable.
Non-primate lentiviruses provide a possible system for the development of new vector systems; however, relatively little is known about these viruses. Although their biology has received considerably less scrutiny than that of the primate lentiviruses (e.g., HIV-1, HIV-2 and SIV), non-primate lentiviruses are of interest for comparative lentivirus biology and as potential sources of safer lentiviral vectors. HIV-based retroviral vectors have recently shown promise for therapeutic gene transfer because they display the lentiretrovirus-specific property of permanently infecting non-dividing cells (see, Naldini et al. (1996) Science 272, 263-267). In contrast, retroviral vectors derived from simpler retroviruses (e.g., the Oncovirinae) require breakdown of the nuclear envelope during mitosis to complete reverse transcription and integration. Consequently, these vectors transduce non-dividing cells poorly, which may limit usefulness for gene transfer to quiescent or post-mitotic cellular targets. However, HIV vectors present complex safety problems (see, Emerman (1996) Nature Biotechnology 14, 943).
The non-primate lentiviruses include the ungulate lentiviruses, including visna/maedi virus, caprine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and bovine immunodeficiency virus (BIV). These lentiviruses only infect hoofed animals (ungulates) and generally only infect particular species of ungulates.
The non-primate lentiviruses also include feline immunodeficiency virus (FIV) (see, Clements and Zink (1996) Clinical Microbiology Reviews 9, 100-117), which only infects felines. Numerous strains of FIV have been identified.
Non-primate (e.g., feline and ungulate) lentiviruses may provide a safer alternative than primate lentiviral vectors, but their use is complicated by a relative lack of knowledge about their molecular properties, especially their adaptability to non-host animal cells (Emerman, id). All lentiviruses display highly restricted tropisms (see, Clements and Zink (1996), supra, and Haase (1994) Annals of the New York Academy of Sciences 724, 75-86).
FIV was discovered in 1986 as a cause of acquired immune deficiency and neurological disease in, and only in, domestic cats (Felis catus) Pedersen et al. (1987) Science 235, 790-793 (1987); Elder and Phillips (1993) Infectious Agents and Disease 2, 361-374; Pedersen (1993) xe2x80x9cThe feline immunodeficiency virusxe2x80x9d in The Retroviridae (ed. Levy, J. A.) 181-228 (Plenum Press, New York Bendinelli et al. (1995) Clinical Microbiology Reviews 8, 87-112; and, Sparger (1993) Veterinary Clinics of North America, Small Animal Practice 23, 173-191). In the great cats, FIV is widely dispersed geographically and appears to be commensal: 18 of 37 species of free-roaming, non-domestic Felidae are known to be infected world-wide, but none develop disease (Elder and Phillips (1993), supra; Olmsted et al. (1992) Journal of Virology 66, 6008-6018; Barr et al. (1995) Journal of Virology 69, 7371-7374; Courchamp and Pontier (1994) xe2x80x9cFeline immunodeficiency virus: an epidemiological review.xe2x80x9d Comptes Rendus de L Academie des Sciences. Serie III, Sciences de la Vie 317, 1123-1134). The virus is prevalent, infecting 2-20% of domestic cat populations in North America, Europe and Japan; higher rates are seen in cats brought to veterinary attention (Pedersen (1993), supra and Courchamp, F. and Pontier (1994), supra). The worldwide prevalence of FIV in diverse Felidae and the observation that Felis catus sera dating to the 1960""s show similar high rates of positivity, suggest that FIV has not been recently introduced into domestic cats (Bendinelli et al. (1995), supra, Olmsted et al. (1992), supra; Courchamp, F. and Pontier (1994) supra; Shelton et al. (1990) Journal of Acquired Immune Deficiency Syndromes 3, 623-630; Bennett and Smyth (1992) British Veterinary Journal 148, 399-412; Brown et al. (1993) Journal of Zoo and Wildlife Medicine 24, 357-364; Carpenter and O""Brien (1995) Current Opinion in Genetics and Development 5, 739-745.
There is no evidence for FIV infection of non-felids. Cross-infection by any of the ungulate or feline lentiviruses has never been observed in non-ungulates, or non-felids respectively. HIV and FIV differ notably in their modes of transmission since FIV is spread principally by biting (Pederson (1993), supra). Despite frequent exposure of humans to FIV through bites by domestic cats, this plausibly efficient means of inoculation does not result in human seroconversion or any other detectable evidence of human infection or disease (Pedersen (1993) id.; Bendinelli et al. (1995) supra.; Sparger (1993), supra; Courchamp, F. and Pontier (1994), supra; Brown et al. (1993).
FIV is also genetically and antigenically distant from the primate lentiviruses. Nucleotide sequence comparisons indicate a closer relationship to the ungulate lentiviruses than to HIV and SIV (Olmsted et al. (1989) Proceedings of the National Academy of Sciences of the United States of America 86, 2448-2452 (Olmsted et al. (1989) A); Olmsted et al. (1989) Proceedings of the National Academy of Sciences of the United States of America 86, 8088-8092 (Olmstead et al. (1989) B). Serological cross reactivity of FIV core proteins to several ungulate lentiviruses has been observed but none occurs to HIV-1, HIV-2 or SIV (Elder, J. H. and Phillips (1993), supra; Bennett, M. and Smyth (1992), supra; Olmsted et al. (1989) A, supra; Talbott et al. (1989) Proceedings of the National Academy of Sciences of the United States of America 86, 5743-5747; Miyazawa et al. (1994) Archives of Virology 134, 221-234). The virus encodes a dUTPase; this fifth pol-encoded enzymatic activity is a feature found only in non-primate lentiviruses (Wagaman et al. (1993) Virology 196, 451-457). Phylogenetic and epidemiologic data suggest an ancient adaptive episode between FIV and ancestors of wild felines as well as early evolutionary divergence from ancestors of other lentiviruses (Olmsted, R. A. et al. (1992), supra; Brown et al. (1993), supra; Carpenter and O""Brien (1995) Talbott et al. (1989), supra.).
At the cellular level, restrictions in human cells to both the productive and infective stages of the non-primate lentivirus life cycles are also evident (Tomonaga et al. (1994) Journal of Veterinary Medical Science 56, 199-201; Miyazawa et al. (1992) Journal of General Virology 73, 1543-1546; Thormar and Sigurdardottir (1962) Acta Pathol Microbiol Scandinav 55, 180-186; Gilden et al. (1981) Archives of Virology 67, 181-185; Carey and Dalziel (1993) British Veterinary Journal 149, 437-454. The bases for these blocks, which might impede development of non-primate lentivirus-based vectors for human application, are not well understood.
The CD4 molecules of primates are the only known lentivirus primary receptors (e.g., for HIV). Neither primary nor secondary receptors have previously been determined for any of the non-primate lentiviruses. Although an antibody that binds to the feline homologue of CD9 inhibits FIV infection in tissue culture (Willett (1994) Immunology 81, 228-233), subsequent investigation has established that neither CD9 nor the feline homologue of CD4 are FIV receptors (Willett et al. (1997) Journal of General Virology 78, 611-618). Reported obstacles to FIV expression in human cells have included poor function of core viral functions such as the Rev/RRE regulatory axis (Tomonaga, K. et al. (1994) supra; Simon et al. (1995) Journal of Virology 69, 4166-4172) and poor promoter activity of the long terminal repeat (LTR) (Miyazawa et al. (1992), supra; Sparger et al. (1992) Virology 187, 165-177. Because of these blocks, expression of the non-primate lentivirus Rev-dependent structural proteins in non-host animal cells has received very limited study.
In addition to the question of restricted tropism, production of non-primate lentivirus vectors in ungulate or feline cells for clinical use would create hazards for transmission of endogenous retroviruses or other potential pathogens. This risk has been documented for cells of diverse animal origin (Stoye and Coffin (1995) Nature Medicine 1, 1100; van der Kuyl et al. (1997) Journal of Virology 71, 3666-3676). Feline cells also contain multiple copies of a replication-competent, type C endogenous retrovirus (RD114) that replicates in human cells, phenotypically mixes with other retroviruses, and is related at the nucleotide sequence level to a primate retrovirus (baboon endogenous virus) (McAllister et al (1972) Nature New Biol 235, 3-6). Moreover, unlike most other type C mammalian retroviruses, RD114 resists inactivation by human serum complement (Takeuchi et al. (1994) Journal of Virology 68, 8001-8007). Cat cells may also contain other unknown and potentially pathogenic infectious agents.
Accordingly, there is a need in the art for safer lentiviral vectors, e.g., for the delivery of genes, cancer therapeutics, viral inhibitors and the like to non-dividing cells, including hematopoietic stem cells and neuronal cells, and for human vector packaging cells capable of packaging non-primate lentiviral vectors. The present invention provides these and other features.
This invention describes retroviral packaging systems, vectors, packagable nucleic acids and other features based on the discovery and design of non-primate lentiviral vectors which are active in human cells. The vectors, which are packageable by a non-primate lentivirus such as FIV, transduce non-dividing human cells. The packaging systems produce vector packaging components in trans in human cells, thereby avoiding the possibility of introducing new pathogens into the human population. These vectors and packaging components are useful for construction of general gene transfer vectors and for human gene therapy.
One class of retroviral vector provided by the invention has a vector nucleic acid packaged by a non-primate letivirus, such as FIV or an ungulate retrovirus. Thus the vector has a packagable nucleic acid which is recognized and packaged by the viral packaging proteins encoded by the selected retrovirus (e.g., FIV). The vector nucleic acid also includes a heterologous target nucleic acid such as a therapeutic gene. The vector nucleic acid is not virulent, because the nucleic acid lacks, or is defective, for one or more gene necessary for viral replication. However, when the missing or defective component (e.g., a retroviral protein) is provided in trans (e.g., in a packaging cell) the vector nucleic acid is packaged in a retroviral viral particle. The vectors optionally include vector packaging or replication elements such as viral proteins, viral particles, reverse transcriptase activity, or the like.
One preferred class of targets for the vectors of the invention are human cells, particularly non-dividing cells such as terminally differentiated hematopoietic cells and neurons (the cells are in vitro or in vivo). Accordingly, features directed to transduction and infection of human cells are preferred features. For example, incorporation of vesicular stomatitis virus (VSV) glycoprotein on the surface of the vector is preferred in some embodiments, as this facilitates entry of the vector into a variety of human cells. Similarly, incorporation of a promoter (e.g., the CMV promoter or a t-RNA promoter) which directs expression of one or more nucleic acid encoded by the vector in a human cell is desirable for production of nucleic acids and, optionally, proteins encoded by the vector in a human target cell.
Packaging plasmids which encode viral components which package the vector nucleic acids in trans are also provided. The packaging plasmids include a promoter which is active in a human cell (i.e., a human cell used for vector packaging). This promoter is operably linked to a nucleic acid encoding at least one protein necessary for packaging the vector nucleic acid, e.g., an FIV or other non-primate lentivirus packaging nucleic acid. The packaging plasmid lacks an FIV packaging site.
In one preferred embodiment, the packaging plasmid has an FIV LTR having a U3 promoter deletion, typically with a heterologous promoter insertion into the deletion site. This arrangement results in eliminating endogenous FIV LTR promoter function and permitting regulation by the heterologous promoter.
It will be appreciated that retroviral packaging cells which package non-primate lentiviral packagable nucleic acids are provided by the packaging plasmids described above. In particular, the cells, which are preferably human, comprise packaging plasmids encoding necessary viral packaging elements (e.g., Gag and Env proteins). These packaging elements are used to package packageable vector nucleic acids in trans. For safety reasons, it is often preferable for the cell to include separate packaging plasmids, each of which encode different packaging proteins. For example, a packaging cell can include two separate packaging plasmids encoding distinct retroviral packaging proteins (e.g., FIV Gag and Env proteins). The cell can further comprise a plasmid which encodes a pseudotyping element such as the VSV envelope glycoprotein to expand the range of any packaged plasmid. The psudotyping element can be encoded on the same plasmid as other non-primate retroviral elements, or on a separate plasmid. In any case, desired packaging elements are under the control of suitable regulatory elements which direct expression of the components in a human cell. It will be appreciated that packageble nucleic acids (e.g., which include an FIV packaging site and a heterologous nucleic acid) are optionally transduced (stably or transiently) into the cells of the invention.